Interleukin-6 enhances insulin secretion by increasing glucagon-like peptide-1 secretion from L cells and alpha cells

Abstract

Exercise, obesity and type 2 diabetes are associated with elevated plasma concentrations of interleukin-6 (IL-6). Glucagon-like peptide-1 (GLP-1) is a hormone that induces insulin secretion. Here we show that administration of IL-6 or elevated IL-6 concentrations in response to exercise stimulate GLP-1 secretion from intestinal L cells and pancreatic alpha cells, improving insulin secretion and glycemia. IL-6 increased GLP-1 production from alpha cells through increased proglucagon (which is encoded by GCG) and prohormone convertase 1/3 expression. In models of type 2 diabetes, the beneficial effects of IL-6 were maintained, and IL-6 neutralization resulted in further elevation of glycemia and reduced pancreatic GLP-1. Hence, IL-6 mediates crosstalk between insulin-sensitive tissues, intestinal L cells and pancreatic islets to adapt to changes in insulin demand. This previously unidentified endocrine loop implicates IL-6 in the regulation of insulin secretion and suggests that drugs modulating this loop may be useful in type 2 diabetes.

Figure 1

Effect of acute IL-6 on GLP-1 and insulin secretion in vivo. (a) Plasma IL-6 (left; resting, n = 3 and running, n = 12) and GLP-1 (right; resting, n = 3 and running, n = 4) in resting and running male mice. Arrow at 90 min indicates exhaustion and blood sampling. (b) Plasma GLP-1 in male mice before and after 90 min of running (n = 6). KO, knockout; IL-6^AB, antibodies to IL-6. (c) Intraperitoneal glucose tolerance test (ipGTT) (left) and oral glucose tolerance test (OGTT) (right) in female mice after a single injection of NaCl (ctrl) or 400 ng of IL-6 (n = 4). IL-6^inj, mice injected with IL-6 .(d) OGTT after a single injection of NaCl or IL-6 in female mice (n = 12). (e) Plasma insulin (left) and GLP-1 (right) concentrations in female mice in response to oral glucose after a single injection of NaCl or IL-6 (n = 4); −30 min indicates the baseline measurement before NaCl or IL-6 injection, and 0 min indicates time point of glucose administration. (f) OGTT in male wild-type (WT) littermates (left) and GLP-1–receptor knockout (Glp1r^−/−) (right) mice after a single injection of NaCl or 400 ng of IL-6 (n = 6–10). (g) Oral-glucose–stimulated insulin secretion in male WT littermate (left) and Glp1r^−/− (right) mice after a single injection of NaCl or 400 ng of IL-6 (n = 6–10). (h) OGTT in male mice after a single injection of NaCl or 400 ng of IL-6 in the absence and presence of exendin (ex) (9–39) (n = 4). Data represent means ± s.e.m. *P < 0.05, determined by Student’s t test comparing control to IL-6 injection, resting to running or IL-6 to IL-6 plus exendin (9–39). *P < 0.05, $P < 0.05, #P < 0.05, determined by analysis of variance (ANOVA) comparing control to IL-6 injections (d,e).

Figure 2

Effect of 1-week IL-6 injections on glucose homeostasis and GLP-1 production. (a) Fasting and fed blood glucose concentrations in male control mice and IL-6^inj mice (n = 8). (b) Fasting plasma hormones in male control and IL-6^inj mice (n = 6–8). (c) Plasma GLP-1 concentrations in response to oral glucose in male control and IL-6^inj mice (n = 8). (d) Intraperitoneal GTT (ipGTT) (left) and plasma insulin in response to intraperitoneal glucose (right) in male control and IL-6^inj mice (n = 8). (e) IpGTT (left) and plasma insulin in response to intraperitoneal glucose (right) in male control mice in the absence or presence of exendin (ex) (9–39) (n = 4). (f) IpGTT (left) and plasma insulin in response to intraperitoneal glucose (right) in male IL-6^inj mice in the absence or presence of exendin (9–39) (n = 4). (g) Intestinal proglucagon (Gcg) mRNA expression (left) and intestinal GLP-1 content (right) in male control and IL-6^inj mice (n = 8). (h) Pancreatic GLP-1 (left), glucagon (middle) and insulin (right) abundance in male control and IL-6^inj mice (n = 8). (i) GLP-1 release over 24 h in isolated mouse islets from male control and IL-6^inj mice (n = 5 PER GROUP). (j) Intestinal mRNA expression in male control and IL-6^inj mice. Data are expressed as a fold of the jejenum control (n = 8). ND, not detectable. Data represent means ± s.e.m. *P < 0.05, determined by Student’s t test comparing control to IL-6^inj mice.

Figure 3

Effects of IL-6 on GLP-1 secretion in GLUTag cells. (a) Western blots of IL-6 receptor (top, with HeLa cell extract as the positive control (ctrl)), pSTAT3 (middle) and actin (bottom). (b) GLP-1 secretion after 15 min of stimulation with IL-6 and 0.1 mM glucose; we used 1 μM glucose-dependent insulinotropic peptide (GIP) as the positive control (n = 5). (c) Capacitance traces following stimulation with IL-6 (top left). Average capacitance following 15 min of stimulation with or without IL-6 (top right, n = 6–11). Ca²⁺ current following stimulation with or without IL-6 (bottom left, n = 6–11). Average capacitance following 15 min of stimulation with or without IL-6 and AG490 (bottom right, n = 5–9). (d) GLP-1 secretion in response to 2 h of stimulation with 11 mM glucose after 24 h with or without IL-6 (left, n = 3). GLP-1 content after 24 h with or without IL-6 (middle, n = 6). GLP-1 secretion in response to 2 h of stimulation with 11 mM glucose after 0–24 h with or without IL-6 (right, n = 3). (e) GLP-1 secretion in response to 2 h of stimulation with 11 mM glucose after 24 h with or without IL-6 (left, n = 3). GLP-1 content after 24 h with or without IL-6 (right, n = 3). (f) GLUTag mRNA in response to IL-6; data are expressed as a fold of the untreated control (broken line) (n = 3–6). (g) GLUTag mRNA after 24 h with or without IL-6 (n = 3). (h) 2-deoxy-³H-D-glucose (³H-2dG) uptake after 24 h with or without IL-6 (n = 4). (i) GLP-1 secretion in response to 2 h of stimulation with 11 mM glucose after 24 h with or without IL-6 (n = 3). (j) GLP-1 secretion in response to 2 h of stimulation with 0.1 or 11 mM glucose after 24 h with or without IL-6 (n = 6). Data represent means ± s.e.m. *P < 0.05, determined by ANOVA.

Figure 3

Effects of IL-6 on GLP-1 secretion in GLUTag cells. (a) Western blots of IL-6 receptor (top, with HeLa cell extract as the positive control (ctrl)), pSTAT3 (middle) and actin (bottom). (b) GLP-1 secretion after 15 min of stimulation with IL-6 and 0.1 mM glucose; we used 1 μM glucose-dependent insulinotropic peptide (GIP) as the positive control (n = 5). (c) Capacitance traces following stimulation with IL-6 (top left). Average capacitance following 15 min of stimulation with or without IL-6 (top right, n = 6–11). Ca²⁺ current following stimulation with or without IL-6 (bottom left, n = 6–11). Average capacitance following 15 min of stimulation with or without IL-6 and AG490 (bottom right, n = 5–9). (d) GLP-1 secretion in response to 2 h of stimulation with 11 mM glucose after 24 h with or without IL-6 (left, n = 3). GLP-1 content after 24 h with or without IL-6 (middle, n = 6). GLP-1 secretion in response to 2 h of stimulation with 11 mM glucose after 0–24 h with or without IL-6 (right, n = 3). (e) GLP-1 secretion in response to 2 h of stimulation with 11 mM glucose after 24 h with or without IL-6 (left, n = 3). GLP-1 content after 24 h with or without IL-6 (right, n = 3). (f) GLUTag mRNA in response to IL-6; data are expressed as a fold of the untreated control (broken line) (n = 3–6). (g) GLUTag mRNA after 24 h with or without IL-6 (n = 3). (h) 2-deoxy-³H-D-glucose (³H-2dG) uptake after 24 h with or without IL-6 (n = 4). (i) GLP-1 secretion in response to 2 h of stimulation with 11 mM glucose after 24 h with or without IL-6 (n = 3). (j) GLP-1 secretion in response to 2 h of stimulation with 0.1 or 11 mM glucose after 24 h with or without IL-6 (n = 6). Data represent means ± s.e.m. *P < 0.05, determined by ANOVA.

Figure 4

Effects of IL-6 on GLP-1 secretion in human islets and human alpha cells. (a) GLP-1 release (left) over 24 h after 4 d with or without IL-6 and with (+) or without (−) the IL-6 receptor antagonist Sant7 (n = 3). GLP-1 secretion (right) in response to 1 h of static incubation in 10 mM arginine after 5 days with or without IL-6 and with (+) or without (−) Sant7 (n = 3). (b) Human islet insulin secretion in response to 1 h of static incubation in unconditioned or conditioned medium from human islets in the absence and presence of exendin (9–39) (n = 3). (c) Basal GLP-1 and IL-6 released from various human islet preparations (n = 6, r² = 0.46, P = 0.058). (d) GLP-1 (left) and glucagon (right) released over consecutive 24 h intervals with or without IL-6 (n = 4). (e) GLP-1 (left) and glucagon (right) secretion in response to 1-h glucose incubations after 4 d with or without IL-6 (n = 4). (f) GLP-1 content (left), glucagon content (middle) and GLP-1:glucagon content as a molar ratio (right) after 4 days with or without IL-6 (n = 3–4). (g) FACS-enriched human alpha cell mRNA in response to IL-6 (n = 3–6). (h) FACS-sorted human beta cell mRNA in response to IL-6 (n = 4). Data represent means ± s.e.m. *P < 0.05, determined by Student’s t test comparing control to IL-6 or unconditioned to conditioned medium, or determined by ANOVA (g,h) comparing control (at time 0) to IL-6.

Figure 5

Effect of acute IL-6 on insulin secretion in animal models of prediabetes and diabetes. (a) Plasma insulin (left) and oral glucose tolerance test (OGTT) (right) in response to oral glucose in male chow-fed mice after a single injection of NaCl (ctrl) or 400 ng of IL-6 (n = 8). (b) Plasma insulin (left) and OGTT (right) in response to oral glucose in male mice fed a high-fat diet (HF) for 18 weeks after a single injection of NaCl or 400 ng of IL-6 (n = 8). (c) Plasma insulin (left) and OGTT (right) in response to oral glucose in male ob/ob mice after a single injection of NaCl or 400 ng of IL-6 (n = 8). (d) Plasma insulin (left) and OGTT (right) in response to oral glucose in male db/db mice after a single injection of NaCl or 400 ng of IL-6 (n = 5). (e) Plasma insulin (left) and OGTT (right) in response to oral glucose in mice fed a high-fat diet and treated with STZafter a single injection of NaCl or 400 ng of IL-6 (n = 5). Data represent means ± s.e.m. *P < 0.05, determined by Student’s t test comparing control to IL-6 injection.

Figure 6

Effect of IL-6 antagonism in mice fed a high-fat diet and in db/db mice. (a) Pancreatic glucagon (left), GLP-1 (middle) and GLP-1:glucagon content as a molar ratio (right) in male WT and IL-6 knockout (KO) mice after 18 weeks of chow or high-fat diet (HF) (n = 8). (b) Immunohistochemistry of pancreatic tissue sections using antibodies against PC1, PC3 and glucagon (representative image of n = 5) (c) Pancreatic glucagon (left) and GLP-1 (right) in male mice fed chow or a high-fat diet for 15 weeks (n = 7–8). Mice on a high-fat diet and injected with IL-6 (HF IL-6^inj) received IL-6 twice daily for the last 7 days of the study, and mice fed on a high-fat diet and injected with an antibody to IL-6 (HF IL-6^AB) received an antibody that neutralized IL-6 for the last 4 weeks of the study. (d) Pcsk1 mRNA in FACS-sorted alpha cells from male mice expressing a yellow fluorescent protein under the control of the glucagon promoter after 20 weeks on chow or a high-fat diet (n = 5–8). Blood glucose (e), ipGTT (left), ITT (middle) and plasma insulin (right) (f) in response to intraperitoneal glucose in male db/db mice after 4 weeks without (db/db ctrl) or with (db/db IL-6^AB) treatment with antibodies to IL-6 (n = 4–5). (g) Fasting plasma hormones in male db/db mice with or without treatment with antibodies to IL-6 (n = 4–5). (h) Pancreatic hormone content in male db/db mice with or without treatment with antibodies to IL-6 (n = 4–5). Data represent means ± s.e.m. *^,#P < 0.05, determined by ANOVA, where the asterisk compares chow to a high-fat diet, and ^# compares genotypes on a high-fat diet only (a) or high-fat diet to a high-fat diet plus antibodies to IL-6 (c). In e–h, *P < 0.05, determined by Student’s t test comparing control mice to mice injected with antibodies to IL-6.

Figure 6

Effect of IL-6 antagonism in mice fed a high-fat diet and in db/db mice. (a) Pancreatic glucagon (left), GLP-1 (middle) and GLP-1:glucagon content as a molar ratio (right) in male WT and IL-6 knockout (KO) mice after 18 weeks of chow or high-fat diet (HF) (n = 8). (b) Immunohistochemistry of pancreatic tissue sections using antibodies against PC1, PC3 and glucagon (representative image of n = 5) (c) Pancreatic glucagon (left) and GLP-1 (right) in male mice fed chow or a high-fat diet for 15 weeks (n = 7–8). Mice on a high-fat diet and injected with IL-6 (HF IL-6^inj) received IL-6 twice daily for the last 7 days of the study, and mice fed on a high-fat diet and injected with an antibody to IL-6 (HF IL-6^AB) received an antibody that neutralized IL-6 for the last 4 weeks of the study. (d) Pcsk1 mRNA in FACS-sorted alpha cells from male mice expressing a yellow fluorescent protein under the control of the glucagon promoter after 20 weeks on chow or a high-fat diet (n = 5–8). Blood glucose (e), ipGTT (left), ITT (middle) and plasma insulin (right) (f) in response to intraperitoneal glucose in male db/db mice after 4 weeks without (db/db ctrl) or with (db/db IL-6^AB) treatment with antibodies to IL-6 (n = 4–5). (g) Fasting plasma hormones in male db/db mice with or without treatment with antibodies to IL-6 (n = 4–5). (h) Pancreatic hormone content in male db/db mice with or without treatment with antibodies to IL-6 (n = 4–5). Data represent means ± s.e.m. *^,#P < 0.05, determined by ANOVA, where the asterisk compares chow to a high-fat diet, and ^# compares genotypes on a high-fat diet only (a) or high-fat diet to a high-fat diet plus antibodies to IL-6 (c). In e–h, *P < 0.05, determined by Student’s t test comparing control mice to mice injected with antibodies to IL-6.